1.1 Generating pluripotent cells from adult mouse and human sources. [unreadable] [unreadable] Our expertise in generating pluripotent cells was demonstrated by the production of a new type of pluripotent cell (EpiS) from the mouse embryo. This cell may represent the epiblast at the time of implantation rather than the pre-implantation inner cell mass that has previously been considered the most likely homologue of ES cells. This new cell is a better model for human ES cells and because it is a later step in development will permit better control of the differentiation of pluripotent cells into distinct somatic cell types. In the last year, our attention has focussed on the production of pluripotent cells from adult tissues. Using different cell types from adult mouse and human tissues, we have generated more than 12 induced pluripotent cells (iPS). The patterns of gene expression in these iPS cells and embryo derived pluripotent cells are remarkably similar. In the next year, we will determine if the same signaling events control the differentiation of iPS, ES and EpiS cells. In this way, we plan to exploit the potential of reprogramming technology to define how genetic change alters signaling and differentiation in human cells. [unreadable] [unreadable] [unreadable] 1.2 Generating pluripotent cell lines from the cat.[unreadable] [unreadable] To date pluripotent embryonic stem cell lines have only been derived in a limited number of species including mouse, chicken, and primate. Our generation of pluripotent cell lines from the mouse epiblast (EpiSCs) may be a step towards a general method to derive ES cells from any vertebrate. We will test this possibility by generating EpiSCs from the cat epiblast and iPS cells from adult cat cells. We initiated work on rat pluripotent cells but with Dr. James Kehler joining the group, an opportunity arose to generate cat pluripotent cells. The cat is an important model in neuroscience and we are making good progress generating iPS cells from cat fibroblasts. [unreadable] [unreadable] [unreadable] 1.3 To define the chromatin state in ES cells and in neural precursors.[unreadable] [unreadable] There is great interest in how the stable epigenetic state of chromatin is maintained in ES cells. The derivation of EpiSCs cells allows a direct comparison of the state of chromatin in two distinct cells that are both pluripoitent. We are using gene array and chromatin immunoprecipitation (ChIP) methods to analyze the epigenetic changes when pluripotent cells differentiate into neural precursors. The differentiation of the cells is controlled by genetic and pharmacological tools. The data show that we have achieved reproducible and robust differentiation of ES cells and this provides a strong basis for a stringent assessment of the differentiation potential of iPS cells.[unreadable] [unreadable] [unreadable] 2.1 Lineages in CNS stem cells.[unreadable] [unreadable] To understand fate specification in neural stem cells we have developed an imaging system to identify every step in the lineage that transforms multipotent cells into astrocytes, oligodendrocytes and neurons. In the standard conditions, tripotent cells produce specified progenitors through bipotent intermediates and, surprisingly, the tripotent state is regenerated from cells with lower potency when cells are passaged. The action of the pro-astrocytic cytokine CNTF demonstrates that the fate specifying events occur rapidly in tri-potent cells. This precise timing of fate specification provides a strong basis for further analysis of the mechanisms that generate specific cell types from stem cells.[unreadable] [unreadable] [unreadable] 2.2 Energy production in neural stem cells[unreadable] [unreadable] It has been proposed that stem cells and their differentiated progeny use different strategies to generate energy. We have shown that neural stem cells and their immediate derivatives use different lactate dehydrogenase genes. In this way, ATP is generated in stem cells by glycolysis and in differentiated cells by oxidative phosphorylation. This finding has important implication in cancer and ischemia.[unreadable] [unreadable] [unreadable] 2.3 An in vitro assay that predicts stem cell activation in vivo.[unreadable] [unreadable] We have shown that Notch ligands stimulate the stem cell survival in vitro and have powerful neurotrophic effects in vivo. Our goal in this project is to determine if in vitro analysis of the survival network predicts the in vivo neurotrophic effect. In the past year, we have shown that insulin and Notch ligands act in a co-operative manner to control stem cell survival in vitro. This result provides an explanbation for a surprising feature of an earlier report from our group. In this report we showed that Notch ligands activate the major growth pathway in a cell. This growth pathway is normally thought to be activated by receptor tyrosine kinases but Notch does not carry this function. By demonstrating that (1) Notch ligands cause tyrosine phosphorylation of the IGFR1 and (2) Notch antagonsists block IGFR1 activation we provide a reciprocal link between Notch and the classic growth pathway in cells. Insulin injection into the adult brain increases stem cell numbers showing that the association between stem cells survival in vitro and in vivo continues to hold. To extend this finding we are developing simple assays that predict the in vivo effects on the stem cell niche. By automatic microscopy, we propose to generate thousands of data points allowing rapid optimization of regenerative therapies.[unreadable] [unreadable] 3.1 To determine neuronal survival mechanisms.[unreadable] [unreadable] To derive and sustain functional neurons it is necessary to understand neuronal survival mechanisms. Using primary hippocampal neurons, we established that young neurons go through a restricted period when they die and neurotrophins are required for their survival. In vitro pharmacological studies suggested that activation of the neurotrophin receptor is only transient and that neuronal survival is achieved by the sustained action of L-type calcium channels and integrins. This work sets up a powerful in vitro assay to study survival signaling in neurons that complements our work in ES cells and CNS stem cells. We have now shown using antibodies and drugs that neurotrophins act through integrins to control the survival of hippocampal pyramidal neurons in vivo. To our knowledge, this is the first definition of the signaling steps controlling the survival of hippocampal neurons in vivo. This work establishes a powerful system to analyze the activity dependant mechanisms controlling neuronal survival in the most intensely studied region of the brain.